Download PDF by Nathan P. Kaplan, Nathan P. Colowick, William B. Jakoby,: Affinity Techniques - Enzyme Purification: Part B

By Nathan P. Kaplan, Nathan P. Colowick, William B. Jakoby, Meir Wilchek

The severely acclaimed laboratory general, Methods in Enzymology, is likely one of the such a lot hugely revered guides within the box of biochemistry. because 1955, every one quantity has been eagerly awaited, often consulted, and praised through researchers and reviewers alike. The sequence comprises a lot fabric nonetheless proper this day - really a vital booklet for researchers in all fields of lifestyles sciences

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5. T N B S Tests. The formation of trinitrophenyl derivatives of primary amines, hydrazides [see Eq. (21)] and hydrazines provides the basis for an extremely useful color test for these groups either when bound to gels or in free solution. In the former case, a small amount of saline-washed gel is suspended in saturated sodium borate in a spotplate well (or test tube). Three to 5 drops of a 3% w/v solution of 2,4,6-trinitrobenzenesulfonic acid (TNBS) in water are added with mixing. Primary amines give an orange color, after several minutes, ranging from yellow to orangered depending on the concentration of groups in the gel.

A°D. A. Rees, Chem. Ind. 16, 630 (1972). 1. 20 COUPLINGREACTIONSAND GENERAL METHODOLOGY [2] Activation step: (~)--OH + X--R--Y *- (~-O--R--Y + HX Coupling step: ( • ) - - O - -+RH2N-Ligand --Y *" (~)--O--R--Y'-Ligand + NHs Activation may also include several steps, as in the case of diazo coupling: (~)--OH + C I C H 2 ~ - N O 2 (~- O-- CH2~-~NO No. < f NH diaz°tizati°n~ ( ~ - O - - C H 2 ~ - N2+ It is important to mention that, after coupling of the desired ligand, reactive Y groups may still be present.

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